血小板TLR4在抗磷脂综合征血栓形成中的作用及机制探讨

Thrombosis is not only the major pathological basis and the most prominent clinical manifestations, but also the primary cause of death in APS. Recent studies suggest that the activation of platelets by antiphospholipid antibodies (aPL) plays a pivotal role in the development of thrombosis in APS, although precise mechanisms remain to be elucidated. Toll-like receptor 4 (TLR4) plays a critical role in regulating platelet function, however, to date, no work has focused on the function of platelet TLR4 in APS thrombosis. Our previous study has demonstrated that TLR4 is involved in the thrombotic complications in a murine model of the APS. Reviewing the references and our previous results, we hypothesize that platelet TLR4 is the key player in APL-induced thrombosis in APS. Different assays including cellular and molecular biology as well as intravital image were used in this study. In vitro, the roles of TLR4 signalling in APL-induced activation of platelets were investigated, the effects of platelet TLR4 on the interactions of APL-activated platelets with endothelium and inflammatory cells were also clarified. Both TLR-4-deficient (TLR4-/-) and wild-type (WT) mice were depleted endogenous platelets, and then platelets isolated from WT or TLR4-/- mice were transfused into the platelet-depleted mice, respectively. The effects of platelet TLR4 on APL-induced activation of endothelium and inflammatory cells, fibrin generation, as well as thrombus formation were observed in in vivo models of APS. This study will provide new knowledge in the understanding of the pathogenic mechanisms of APS, and develope new therapeutic modalities to treat and/or prevent thrombosis in APS patients.

血栓形成是抗磷脂综合征（APS）最主要的病理基础和临床表现，也是造成患者死亡的重要原因。近期研究提示抗磷脂抗体（APL）激活血小板是APS血栓形成的首要因素，其具体机制有待阐明。TLR4在调节血小板功能中起重要作用，但血小板TLR4与APS血栓形成的关系尚无报道。我们前期研究表明TLR4参与APS模型小鼠血栓形成。基于文献及前期工作，我们提出血小板TLR4是APS血栓形成的关键。我们拟利用分子生物学、细胞生物学及活体成像等技术，体外探讨TLR4及其信号通路对APL活化血小板的影响，阐明APL诱导的血小板活化对内皮细胞和炎症细胞的影响及血小板TLR4在其中的作用；对血小板耗竭的野生型和TLR4-/-小鼠重新输注野生型或TLR4-/-血小板并建立APS模型，体内证实血小板TLR4对内皮细胞和炎症细胞活化、纤维蛋白产生及血栓形成的影响。本研究将为APS血栓形成提供新视角，为血栓防治提供新思路。

抗磷脂综合征（antiphospholipid syndrome，APS）是一种以反复动、静脉血栓、习惯性流产和血小板减少等为主要临床表现的自身免疫性疾病，抗磷脂抗体（APL）尤其是抗β2GPI抗体能与其抗原β2GPI形成复合物，激活靶细胞发挥促血栓作。血栓形成是APS主要的病理基础，也是导致患者死亡的重要原因。近期研究提示APL激活血小板是APS血栓形成的首要因素，其具体机制有待阐明。近年来TLR4与APS的关系正受到愈来愈多的关注。许多研究发现血小板亦可表达具有生物活性的TLR4，TLR4在调节血小板功能中起重要作用，但血小板TLR4与APS血栓形成的关系尚无报道。基于文献及前期工作，我们提出血小板TLR4是APS血栓形成的关键。我们利用分子生物学、细胞生物学及活体成像等技术，体外探讨TLR4及其信号通路对APL活化血小板的影响，阐明APL诱导的血小板活化对内皮细胞和炎症细胞的影响及血小板TLR4在其中的作用；对血小板耗竭的野生型和TLR4-/-小鼠重新输注野生型或TLR4-/-血小板并建立APS模型，体内证实血小板TLR4对内皮细胞活化及血栓形成的影响。我们的研究结果表明anti-β2GPI/β2GPI复合物可以激活血小板释放P-选择素、sCD40L和β-TG，促进下游信号通路p38-MAPK和NF-κB活化，诱导血小板聚集和对内皮细胞的粘附，TLR4抑制剂TAK-242可以显著减少anti-β2GPI/β2GPI复合物诱导的血小板活化、聚集和粘附。活化的血小板可以激活内皮细胞表达ICAM-1、MCP-1、IL8、IL6、IL1β，促进单核细胞表达TF、TNF-α、IL-1β和IL6，以及诱导中性粒细胞形成NETs。当血小板TLR4被抑制后，血小板对内皮细胞和炎症细胞的刺激效果明显减弱。体内实验显示血小板TLR4缺陷可以显著抑制anti-β2GPI诱导的小鼠颈动脉血栓形成。此外，我们还发现TLR4、PAD4在APL诱导的中性粒细胞胞外诱捕网形成中发挥重要作用。本研究以血小板TLR4为切入点，为APS血栓形成提供新视角，为血栓防治提供新思路。